Histone acetylation by ethanol:Mechanisms & consequence

描述（由申请人提供）： 组蛋白的翻译后修饰是转录激活和基因沉默的重要机制。我们最近进行了最初的观察，乙醇优先引起核组蛋白H3在赖氨酸9处乙酰化的惊人（8-10倍）增加，并且这种增加对低至5 mM的乙醇非常敏感。我们的长期目标是解决如何以及为什么这种乙酰化发生？该项目将奠定基础。我们的假设是：“乙醇在体内和体外都能引起组蛋白的选择性乙酰化，从而影响转录机制。“来自体内乙醇喂养大鼠的肝细胞和肝脏的原代培养物将作为本项目的主要模型。旨在为该项目的未来发展建立框架，有三个目标。 目的研究乙醇诱导肝细胞组蛋白乙酰化的特点。在这一目标下，将研究这种影响的时间过程及其可逆性。我们还将探讨这种乙醇诱导的H3乙酰化反应在不同组织来源的细胞中的特异性和多样性。 目的二是探讨肝细胞组蛋白乙酰化的机制。该方案将检查乙醇代谢抑制剂的作用，个别乙醇代谢产物或衍生成分，以及乙酰辅酶A水平变化在乙酰化反应中的作用。该目的还将解决是否涉及组蛋白乙酰转移酶（HAT）的表达和/或活化增加或组蛋白脱乙酰酶（HDAC）的抑制。 目的III致力于在体内研究，其中液体饮食乙醇喂养对大鼠肝脏H3乙酰化的影响将被确定。将进行使用染色质免疫沉淀（CHIP）测定研究乙酰化对基因表达的后果的实验，这些实验将成为该项目未来扩展的基础。 这个探索性的R21建议涉及酒精领域的一个新方向，并提供了在转录水平上的乙醇效应的机制数据的承诺。此外，参与组蛋白修饰的“机制”将作为一个新的目标，以开发用于预防和治疗乙醇诱导的肝脏和受乙醇影响的其他器官系统中的细胞损伤的治疗工具。

DESCRIPTION (provided by applicant): Post-translational modifications in histones are emerging as important mechanisms for the transcriptional activation and gene silencing. We have recently made the original observation that ethanol preferentially caused a spectacular (8-10 fold) increase in acetylation of nuclear histone H3 at Lys 9 and that this increase was remarkably sensitive to as low as 5 mM ethanol. Our long-term goal is to address how and why this acetylation occurs? This project will lay the foundation. Our hypothesis is that "ethanol causes selective acetylation in histones, both in vitro and in vivo, and that this would consequently affect the transcriptional machinery." Primary cultures of hepatocytes and livers from in vivo ethanol fed rats will serve as the main model in this project. There are three aims designed to build the framework for future development of this project. Aim I will determine the characteristics of ethanol induced histone acetylation in hepatocytes. In this aim, time course of this effect and its reversibility will be studied. We will also explore the specificity and diversity of this ethanol induced H3 acetylation response in cells of different tissue origins. Aim II will determine the mechanism(s) of histone acetylation in hepatocytes. The protocols will examine the effects of inhibitors of ethanol metabolism, individual ethanol metabolites or derived components, and the role of changes in levels of acetyl CoA in the acetylation response. This aim will also address whether increased expression and/or activation of histone acetyl transferase (HAT) or an inhibition of histone deacetylase (HDAC) are involved. Aim III is devoted to in vivo studies where effect of liquid diet ethanol feeding on rat liver H3 acetylation will be established. Experiments to study the consequence of acetylation on gene expression using chromatin immunoprecipitation (CHIP) assay will be performed and these experiments will form the basis for future extension of this project. This exploratory R21 proposal deals with a new direction in the alcohol field and offers the promise of providing mechanistic data into the ethanol effects at transcriptional level. Furthermore, "machinery" involved in histone modification will serve as a new target to develop therapeutic tools for prevention and treatment of ethanol induced cellular damage in liver and other organ systems affected by ethanol.